Cytokinins are small molecules produced by plants that regulate growth and development processes under normal growing conditions as well as under stress conditions. It is well established that cytokinins stimulate growth and differentiation of cultured plant cells. Cytokinin signaling has been shown to correlate with production of tissues during bud outgrowth and root nodule formation. Faiss et al., Plant J., 12(2): 401-15 (1997); Ferguson et al., Plant Physiol., 149(4): 1929-1944 (2009), Tirichine et al., Science, 315(5808):104-107 (2007) and Held et al., Plant Cell, 26(2): 678-694 (2014). Cytokinin inactivation during abiotic stress, e.g., due to drought, cold, or excess salt leads to decreased plant productivity, reduced seed size, increased tip kernel abortion and decreased seed set. (Jones and Setter, in CSSA Special Publication No. 29, pp. 25-42. American Society of Agronomy, Madison, Wis. (1999)). Transgenic expression of cytokinin biosynthesis enzymes has been reported to increase plant productivity, including under abiotic stress. Rivero et al., Proc. Natl. Acad. Sciences USA, 104(49) 19631-36 (2007); Merewitz et al., J. Exp. Bot., 63(3): 1315-1328 (2012). Additionally, formulations of cytokinin derived from algae and other formulations of cytokinin (benzyl adenine) have been registered and approved for uses that include application to field crops, vegetable crops, small fruits, vines, tree fruit, young trees, ornamentals, and golf courses to increase fruit size, yield, blossoms, branching, healthy appearance, and other desirable growth effects. U.S. Environmental Protection Agency Registration Eligibility Decision (RD), EPA-738-R-95-025, December 1995 and N-6 benzyladenine Registration Review Case 2040 (PC Code 116901) March 2011. In plant leaves, foliar applications of exogenous cytokinin (benzyl adenine) have been used to reverse the effects of drying roots, which can be sustained by repeated applications and leads to development of lateral shoots. Stoll et al. J. Exp. Bot., 51(350): 1627-1634 (2000).
The biosynthesis of cytokinins in plants is complex and involves a primary and secondary pathway. Motkya et al., Plant Physiol. 112: 1035-1043 (1996). In the primary pathway, cytokinins are synthesized de novo in a multiple step reaction that begins with the activity of adenosine phosphate-isopentenyltransferases (IPTs), which preferably catalyzes the condensation of adenosine diphosphate (ADP) or adenosine triphosphate (ATP) with prenyl donors to form iP riboside 5′-diphosphate (iPRDP) or iP riboside 5′-triphosphate (iPRTP), respectively. These precursors are either hydrolyzed by cytokinin nucleotide phosphoribohydrolases to form the cytokinin N6-(Δ2-isopentenyl)adenine (iP) or, alternatively, they are converted to corresponding trans-zeatin nucleotides (tZN), which are then hydrolyzed to form the cytokinin trans-zeatin (tZ). In the secondary pathway, cytokinins are generated by degradation of transfer RNA (tRNA). The first step of the pathway involves tRNA-isopentenyltransferase (tRNA-IPT) enzyme that post-transcriptionally modifies tRNA to make prenylated tRNA. This precursor is further modified and condensed with adenine to generate cis-zeatin riboside (cZR) which is hydrolyzed to generate primarily cis-zeatin. Generally, the cytokinin pool produced by the primary pathway is understood to be biologically more active.
The infectious crown gall forming bacterium Agrobacterium tumefaciens has two genes which encode IPTs that preferentially catalyzes the condensation of adenosine monophosphate (AMP) with hydroxymethylbutenyl diphosphate (HMBDP) or dimethylallyl diphosphate (DMAPP) to form trans-zeatin ribosyl monophosphate (tZRMP) which is subsequently hydrolyzed by cytokinin nucleotide phosphoribohydrolases to form trans-zeatin (tZ). Thus, the mechanism of cytokinin biosynthesis in Agrobacterium involves at least two enzymes and produces predominantly tZ cytokinin.
There is a desire for new compositions and methods that can be used to produce or regulate the production of cytokinin in vitro or in vivo. For example, there is a desire for compositions and methods that use a single polypeptide to produce cytokinin directly from precursor. These can be less dependent on the presence of additional upstream or downstream effectors for the production of cytokinins. Such compositions and methods can be used to produce cytokinin formulations for applications to plants. Additionally such can be used to modulate cytokinin production in plants and thereby regulate plant growth and development.